In Vitro Culture And Plantlet Regeneration Of Slash Pine And Loblolly Pine

Slash pine and loblolly pine have become main economic species in southern China since they were introduced in 1930'. The lagging breeding technology for elite clones is limiting the development of the two species. Using various kinds of materials as explants, direct organogenesis, indirect organogenesis and direct somatic embryogenesis of the two species were studied in order to establish a stable and efficient system of plantlet regeneration for slash pine and loblolly pine, which provides reference for tissue culture and rapid breeding of other conifer species. The content and results are as followed:Direct organogenesis and plantlet regeneration of slash pineWith the mature zygotic embryos of antidisease families and non-antidisease families of slash pine as explants, the key technique and main factors affecting vitro culture multiplication of slash pine were studied based on different basic media, hormone compositions in different concentration ratio as well as different plant growth regulators, The results showed that the families or genotypes, the generation frequency and the combination of the different levels of different plant growth regulators were main effect factors. The best medium for multiplication is B5+ 6-BA2.0mg/L+IBA0.1mg/L,in which annual multiplicative rate was 3.696.2.Based on significant difference in buds multiplicative rate among different families or clones,132 elite genotypes in non-antidisease families and 2 elite genotypes in antidisease families of slash pine had been chosed. The conflict between multiplicative rate and the percentage of effective shoots had been resolved through solid culture on two layers of media including a layer of liquid medium with nutrient, meanwhile, the multiplicative culture cycle had been shorten and the culture procedure had been simplified.In the stage of root initiation, the effect of concentration of major element on rooting of shoots were studied. The results showed that the medium without major element benefited rooting of shoots in the beginning of root initiation, the rooting cycle had been shorten and rooting rate had been improved with the protocol that major element were supplemented 25 days after root initiation.The tissue culture technique and rapid breeding of slash pine had been improved and the stable multiplicative and rooting system had been established . Indirect organogenesis and plantlet regeneration of slash pineWith the mature zygotic embryos, sprouts, hypocotyls and radicle of slash pine as explants, the three media DCR, LP, TX combining with different concentration of NAA, 2,4-D, 6-BA were used, the key technique for initiation, multiplication and differentiation of organogenic calli were studied. The results showed that the mature zygotic embryos and sprouts were best explants for initiation of organogenic calli, the best initiation medium was TX+BA 0.5mg/L+NAA 2.0mg/L+30g/L sucrose, with initiation rate reaching 50%; the best multiplication medium was TX+BA 0.3 mg/L+NAA 1.0mg/L+CH 400mg/L+30g/L sucrose, the multiplicative rate was 3.46; the differentiation medium was TX+BA 0.5 mg/L+IBA 0.5 mg/L+30 g/L sucrose, in which more than 20 buds were induced from one callus; the synchronous development medium were TX+BA 0～0.2mg/L+IBA 0.5mg/L+30g/L sucrose. NAA was necessary for initiation and multiplication of organogenic calli. Although the procotol of indirect organogenesis is complicate, the multiplicate frequency in which was much more high than that of indirect organogenesis.The result of study on histology and cytology showed that outer morphology with white, semi-firm and interior structure with large number of meristem is the symbol of OC. Differentiation and development of adventitious buds needed 40-45 days.Somatic embryogenesis of slash pineWith the mature and immature zygotic embryos as explants, the main factors affecting initiation, multiplication and maturation of somatic embryos were studied. The results showed that zygotic embryos with polyembryony are best explants, when zygotic embryos are in development stage 2, 3, 4. There was significant difference in ESM initiation rate among different basic media, different hormone combination. The best ESM initiation media are LP+2,4-D 2.2 mg/L +6-BA 1.0mg/L and DCR+NAA2.0 mg/L +6-BA0.63 mg/L+KT0.61mg/L,in which the initiation rate of 4 families are 24% and 22.75%, respectively. Cold treatment, incubation methods and AgNO3 and MES improved ESM initiation rate.The loss of ESM is common in the stage of ESM maintenance in somatic embryogenesis of conifer. The 4 embryogenic cell line with rapid propagation had been established through optimization of media and hormone combination.The best proliferation medium was DCR+NAA 1.0 mg/L +6-BA 0.32 mg/L + KT 0.30 mg/L.Growth hormone was necessary for maintenance of polarity structure of embryos,but high concentration of growth hormone was harm to the development and maturation of embryos.High concentration of 2,4-D induced unnormal embryos especially.Pretreatment in medium supplemented with AC is necessary.The combination with ABA,PEG and high concentration sucrose improved maturation of embryos. Somatic embryos of stage 5 had achieved in PL+ABA 50 mg/L+PEG6000 20%+6%.Somatic embryogenesis of lobloly pine With immature zygotic embryos from the different cone collection time of elite loblolly pine families 20-692,20-615,20-1036,20-639 as explants , the four patent media AL,TX,LP,DS suitable for somatic embryogenesis combine with their hormone species and levels and without hormone to form 16 initiation media, the influence of different patent media ,different families were studied, different cone collection time on somatic embryos initiation rate and quality. The results of experiment showed that the best explants for somatic embryogenesis wss immature zygotic embryos of development stage 2, 3 and 4. Genotype of explants was main factor affecting initiation rate of somatic embryo and optimization of media and combination of hormone were effective methods for breaking the limit of recalcitration genotype. High initiaton and high quality of somatic embryos had achieved in LP+2,4-D 2.2 mg/L +6-BA 1.0mg/L and AL+2,4-D 2.2 mg/L +6-BA 1.0mg/L.Different ECL had different response to maturation media. Intact somatic embryos developed further when transplanted to maturation media. ECL AL-225 of loblolly pine developed further when precultured on the hormone free maturation medium AL+ABA 50 mg/L+15%PEG8000, but no mature embryo occurred. The sustainable proliferation ESM was obtained from the mature zygotic embryos. The initiation rate of the mature zygotic embryos was 3%, much lower than that of the immature ones, but the somatic embryogenesis of the mature zygotic embryos is important for scientific study.